Otitis media (OM) is the most common childhood bacterial infection and also the leading cause of conductive hearing loss in children. Inflammation is a hallmark of OM. Developing highly effective vaccines still remains a great challenge. Inappropriate antibiotic treatment increased antibiotic resistance substantially. Currently, there are no effective therapeutics including anti-inflammatory agents available for OM due to poor understanding of the pathogenic mechanism underlying inflammation and the effective therapeutic targets. Although appropriate inflammation is essential for eradicating pathogens, excessive inflammation is clearly detrimental to the host. Thus, inflammation must be tightly regulated. However, how inflammation is tightly regulated remains unknown. Our long-term objective is to understand the molecular mechanisms by which inflammation is tightly regulated in OM and to identify effective therapeutic targets. During previous grant period, we identified a novel deubiquitinase CYLD as the key negative regulator for tightly controlling NTHi bacteria-induced inflammation in OM. In view of the past effort in developing anti-inflammatory agents, most strategies have focused on directly targeting the positive pathways, e.g., the I?B kinase (IKK), to suppress inflammation. While these agents often showed reasonable efficacy, they did exhibit significant adverse effects, e.g., increased susceptibility to infection and apoptosis, which prevented their further clinical use. Thus, we hypothesized that up-regulating CYLD, the key negative regulator of inflammation, by pharmacological inhibition of its own negative regulator, may represent a novel and advantageous anti-inflammatory strategy for treating overactive inflammation in OM without causing serious adverse effects often seen with targeting positive regulator of inflammation (hypothesis & innovation). Indeed, our preliminary studies demonstrate that CYLD itself is further negatively controlled by PDE4; Pharmacological inhibition of PDE4 led to the suppression of inflammation by up-regulating CYLD expression likely via JNK. These preliminary data have thus laid a solid foundation for us to investigate the molecular mechanisms underlying negative regulation of CYLD by PDE4B-JNK2 pathway and evaluate the therapeutic effect of up-regulating CYLD by inhibiting its own negative regulator in NTHi-induced inflammation in OM (short-term objective). Our specific aims are: Aim 1. Determine if PDE4B is critical for mediating NTHi-induced inflammation by inhibiting CYLD in vitro & in vivo. Aim 2. Determine how PDE4B regulates NTHi-induced inflammation by inhibiting CYLD via selective activation of JNK2 in vitro & in vivo. Aim 3. Determine the therapeutic effect of PDE4B inhibition on NTHi-induced inflammation in a mouse OM model. Overall, the proposed studies will advance our understanding of molecular pathogenesis of OM and lead to identifying novel therapeutic targets and new anti-inflammatory agents for OM. PUBLIC HEALTH RELEVANCE: - Relevance to Public Health Otitis media (OM), inflammation of middle ear, is the most common childhood bacterial infection, accounting for 24.5 million visits and costing over $5 billion annually, and is also the leading cause of conductive hearing loss in children. Developing highly effective OM vaccines still remains a great challenge, inappropriate antibiotic treatment increased antibiotic resistance substantially and currently there are no effective therapeutics including anti-inflammatory agents available for OM due to poor understanding of the pathogenic mechanism underlying inflammation and the effective therapeutic targets. The currently proposed studies will advance our understanding of molecular pathogenesis of OM and lead to the identification of novel therapeutic targets and development of new anti-inflammatory therapeutic strategy for inhibiting inflammation in OM.